MS-based footprinting reveals that severe WR mutants induce the shift of the VKOR active site to an aberrantly reduced state. (A) Scheme of MS-based footprinting. VKOR cysteines located in the ER are in equilibrium between oxidized and reduced states, and those in the cytosol or inside the ER membrane remain reduced. The membrane permeable NEM (ie, NEM-d₀) is used to label all reduced cysteines in cells. After cell lysis, the oxidized cysteines are subsequently reduced by tris(2-carboxyethyl)phosphine hydrochloride (TCEP), labeled by the NEM-d₅ isotopologue, resulting in a mixture of d₀ and d₅ proteins, and digested. MS quantification to provide the ratio of the isotopically labeled peptides gives the redox state of each cysteine in the cells. (B) Representative product-ion (MS/MS) spectrum of a peptide with both Cys132 and Cys135 labeled by NEM-d₅. (C) Relative reduced levels of cysteines in WT VKOR and severe WR mutants. All 3 severe WR mutants shift Cys51 and Cys132 to an aberrantly reduced state. As controls, Cys16 (TM1), Cys85 (TM2), and Cys96 (cytosolic loop) remain in the reduced state in WT and mutants. The error bars are from 3 different peptides and two-tailed student t test are used. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. (D) Relative redox fractions of cysteine pairs. The severe WR mutants shift Cys132/Cys135 at the active site to an aberrantly reduced state. (E) Structural interpretation of MS detected the state of VKOR cysteines. Severe WR mutants shift the cellular state of Cys132/Cys135 from PO or O state to R state. WT, wild-type.